Supplementary Materials
Supplementary Methods
qPCR Methods
Reactions were performed according to the manufacturer’s instructions (Applied Biosystems, 2009).
Standard curves were constructed using known quantities of genomic DNA with the ABI RNaseP
assay used as a control.
qPCR reactions were performed in quadruplicate on a LightCycler® 480 Real-Time PCR System (Roche
Applied Sciences, USA).
Data analysis was performed using protocols developed within the
AstraZeneca laboratories to determine the relative amounts and standard deviations against the
control gene RNaseP and the genes of interest for each DNA sample.
PCR Methods
Primers were tagged at the 5’ end with M13 forward and M13 reverse oligonucleotide tags
respectively for sequencing purposes. Amplification was carried out in 1.5mM Reddyload™ buffer
(Thermo Scientific) containing 50ng genomic DNA and 200µM of the gene specific primers in a total
volume of 20µl under the following conditions: a single cycle of 94oC for 10 minutes, followed by 14
cycles of 94 oC for 30 seconds, 63 oC (reducing by 0.5 oC/cycle) for 30 seconds followed by 72 oC for 60
seconds, then 25 cycles of 94 oC for 30 seconds, 56 oC for 30 seconds, 72 oC for 60 seconds with a
final single cycle of 72oC for 10 minutes.
Amplification was verified by gel electrophoresis.
Sequencing was carried out using BigDye™ Dye terminator sequencing (Applied Biosystems) on an
ABI 3730™ using oligonucleotides specific to the M13 forward and M13 reverse tags (Table 3).
Sequence data analysis was performed using Phred/Phrap/Consed (CodonCode Corporation, US).
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Amplification of products to delineate the breakpoints of confirmed CNVs was performed using
primers described in supplementary table 3. PCR was performed as follows; 125ng of DNA under the
following conditions: an initial denaturation step of 94oC for 2 minutes followed by 9 cycles of
denaturation at 94oC for 10 seconds, 60oC for 30 seconds and 68oC for 8 minutes, followed by 19
cycles of 94oC for 10 seconds, 60oC for 30 seconds and 68oC for 8 minutes with an increase in
extension time of 10 seconds per cycle.
2
Supplementary Tables
Gene
Region
Assay I.D.
KCNQ1
Exon 13 (upstream)
Hs03783454_cn
KCNQ1
Exon 13 (downstream)
Hs03767069_cn
KCNH2
3’UTR
Hs00126416_cn
Supplementary Table 1. Commercially available quantitative PCR Assays.
Gene
Region
Forward 5’ -3’
Reverse 5’ - 3’
Reporter 1
KCNQ1
15
GGCCTGCGGTGGTTCT
TGCAGGAGCTTCACGTTCA
CACCTCCCTGTGGTCTGCGTG
KCNE1
5’UTR
AGCCTGCTGCCAATGGT
CTTCTCCTACCAGTTCTCGTTTCTG
AAGTCTCCTCAAGCACACTGCG
Supplementary Table 2. Custom made qPCR Assays.
Forward 5’ -3’
Reverse 5’ - 3’
KCNQ1 exon 13
GACACTGTCACTGCCTGCAC
CACCGCTGATCATGCAGCTGG
KCNQ1 exon 15
AGAGGTGGAGAGCGTGGAG
ACTCAGGACACAGCCAGGAG
M13 tags
ACTGTAAAACGACGGCCAGT
ACCAGGAAACAGCTATGACC
Exon 12-15 Long PCR
TCGACGCATGCAGTACTTTGTGGCCAAG
CCTACCTTGTCTTCTACTCGGTTCAGG
Intron 12 (sequencing primer)
TGGCTAGTGTTGCCAGTGAGG
-
Exon 15 (sequencing primer)
-
TTGTCTTCTACTCGGTTCAGG
Supplementary Table 3. Sequencing oligonucleotide sequences.
KQ1-F-NheI
5’-ATATGCTAGCATGGCCGCGGCCTCCTCCCCGC-3’
KQ1mut-R-XmaI
5’ATCTCCCGGGTCAAGCTGGCGCGGATCTAGTGGCTGCC
GCGATTCGTGCACGCTTCTGGAATTTCTTCTTGGCCACA-3’
Supplementary Table 4. Construct PCR Primers. The bold region of the reverse primer is the region
that binds the wild type KCNQ1 channel.
3
Supplementary Figures
Supplementary Fig 1. qPCR validation of CNV MLPA results. Graphs show the ratio of test qPCR
assay against the control gene RNaseP. Copy number loss is indicated by a ratio of < 0.6 and copy
number gain by a ratio of > 1.4.
Panel A Confirmation of single copy gain of KCNE1 in HVP1 by qPCR.
Panel B Single copy loss of KCNQ1x15 in HVP2 was not confirmed by qPCR.
Panel C Confirmation of single copy loss of KCNH2 in aLQT1.
Single copy loss of exon 13 in aLQT2 was confirmed using assays upstream (Panel D) and
downstream (Panel E) of the exon 13 MLPA probe.
A
B
C
D
E
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Supplementary Fig. 2. Results for KCNQ1 copy number variant which failed to be validated in
sample HVP2
Panel A MLPA results for healthy volunteer HVP2 which demonstrate a putative single copy loss of
KCNQ1 exon 15
Panel B. Sequence identification of SNP rs17221854 15bp upstream of the ligation site for the
KCNQ1 exon 15 probe in HVP2.
A
B
rs17221854
Ligation Site
Supplementary Fig 3. DNA quality verses MLPA Success for the aLQT panel
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Supplementary Fig 4. Confirmation and delineation of breakpoints of KCNQ1del531-676 in aLQT2.
Panel A. Sequencing data showing heterozygous break points KCNQ1del531-676
Panel B. Diagrammatic representation of the KCNQ1 gene structure indicating the position of the
deletion and distances in base pairs with respect to the affected exons.
A
Forward Sequence
Reverse Sequence
WT
Sample
aLQT2
B
6218bp
822bp
Exon 12
130bp
2702bp
Exon 13
Exon 14
6348bp
Exon 12
Exon 15
6
813bp
Exon 15